CN112940992A - Tea tree endophytic bacteria with plant growth promoting effect, microbial inoculum and application thereof - Google Patents

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to tea tree endophytic bacteria with a plant growth promoting effect, a microbial inoculum and application thereof. The tea tree endophytic bacteria is Erwinia rhapontici (Erwinia rhapontici) V4, and the strain is preserved in China Center for Type Culture Collection (CCTCC) at 1 month and 7 days 2021, with the preservation number of CCTCC NO: m2021027. The tea tree endophytic bacteria have IAA production capacity, ACC deaminase activity, nitrogen fixation capacity, phosphorus dissolving capacity and siderophore production capacity, so that soil nutrient components and absorption conditions are improved, plants are promoted to absorb more nutrients, and the growth of the plants is effectively promoted. Moreover, the tea tree endophytic bacteria are easy to culture, high in propagation speed, green, organic and pollution-free, and can be widely applied to preparation of plant growth promoting microbial agents or microbial fertilizers.

Description

Tea tree endophytic bacteria with plant growth promoting effect, microbial inoculum and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to tea tree endophytic bacteria with a plant growth promoting effect, a microbial inoculum and application thereof.

Background

At present, pesticides and fertilizers are widely applied in agriculture, and can promote the growth and the high yield of plants. However, with the continuous use of chemical fertilizers, pesticide residues exist in soil and food, and threaten human health and living environment. Therefore, it is a particular concern of people to find efficient and healthy plant growth promoters, which are capable of promoting ineffective nutrition in soil, controlling crop diseases, reducing the use of pesticides and fertilizers, and promoting the growth and development of plants.

In the current research, the plant growth promoting strains are mainly separated from plants or the environment, are applied to soil as microbial fertilizers and microbial inoculants, and have the characteristics of safety, strong adaptability and durability. The Chinese patent application CN104805045A discloses a rhizosphere growth-promoting bacterium and application thereof, the strain has the growth-promoting characteristics of nitrogen fixation, phosphorus dissolution and indoleacetic acid production, can increase soil nutrients, improve the effectiveness of fertilizer, and promote the absorption of soil nutrients by plants, and has obvious growth-promoting effect on wheat. Chinese invention patent CN109136137B discloses a heavy metal resistant plant growth promoting strain and application thereof, the plant growth promoting strain BHG-02 can secrete organic acid, dissolve and utilize calcium phosphate in a culture medium, and has the characteristics of ACC deaminase activity, growth hormone IAA, urease activity and the like, dissolves heavy metals in soil, relieves heavy metal stress of plants, improves soil nutrient condition, and plays a role in promoting plant growth. Chinese patent application CN110484470A discloses a multi-effect plant growth-promoting bacterium and a separation and screening method, wherein the multi-effect plant growth-promoting bacterium has the functions of dissolving phosphorus and producing NH₃The product can produce plant growth hormone and siderophore, and can be used in fertilizer and microbe inoculator.

Tea trees are important economic crops in China, seven things including firewood, rice, oil, salt, sauce, vinegar and tea are opened from ancient times, and the importance of the tea in our lives can be seen. Therefore, the quality of tea is gradually valued by people, and people tend to select tea leaves which are pollution-free food tea, green food tea and organic tea, namely healthy tea without pesticide and chemical fertilizer residues. Therefore, the microbial fertilizer and the microbial inoculum are used for replacing the existing artificially synthesized chemical fertilizer and pesticide, so that the growth, development and yield of the tea trees can be promoted, the organic health of the tea leaves can be ensured, and the method is an important prospect for tea tree planting.

Xiaomei et al (Yan, x., Wang, z., Mei, y., Wang, l., Wang, x., Xu, q., Wei, C. (2018) Isolation, inversion, and Growth-Promoting Activities of Endophytic Bacteria From Tea cultures of zijuan and yunkang-10.Front Microbiol, 9, 1848) isolated Endophytic Bacteria From two closely related Tea varieties of the purple-silk and cloud-resistant 10, compared their Diversity and Plant Growth Promoting (PGP) activity, determined the dominant population of the two varieties or the plant Growth Promoting activity of the nitrogen-fixing genus. The research team obtained 110 isolates of a total of 18 genera of three phyla (proteus, sclerothrix and bacteroides) from Zijuan; 164 isolates of 22 genera of two phyla (proteobacteria and firmicutes) were obtained from Yunzan No. 10. This study showed that the strains of the genus Spirosoma (Herbaspirillum spp.), Methylobacillus (Methylobacillus spp.), Brevundimonas (Brevundimonas spp.) exhibited different plant growth promoting abilities, which were decreased in the order of the strains of the genus Spirosoma, Brevundimonas and Methylobacillus, and most of the Methylobacillus strains did not have plant growth promoting properties of nitrogen fixation, phosphate solubilization, siderophore production, indoleacetic acid production and ACC deaminase activity. In addition, this study demonstrated that endophytes in tea plant varied constantly in different seasons and different tea plant varieties, with the plant-promoting bacteria of Zijuan being much more abundant than Yunkan No. 10. However, although the research relates to the separation and identification of various tea tree endophytic bacteria and the comparison of plant growth promoting capacity, the growth promoting application and the effect verification of the tea tree endophytic bacteria in specific plants cannot be realized.

Erwinia flavum (Erwinia rhapontici) is a gram-negative rod-shaped bacterium, which has been used in the preparation of isomaltulose, for example, Chinese patent CN101200750B, which discloses an Erwinia flavum and its use in the preparation of isomaltulose. However, the reports of the Erwinia rhapontici in the plants are few, and particularly, the related research of the Erwinia rhapontici in the tea plant and the report of the application of the Erwinia rhapontici in promoting the growth of the plants are not available at present.

Disclosure of Invention

The invention aims to provide tea tree endophytic bacteria with a plant growth promoting effect, which can effectively promote the growth of plants, can be applied to the preparation of plant growth promoting bactericides or microbial fertilizers, and is green, organic and pollution-free.

To achieve the above object, a first aspect of the present invention provides a tea tree endophytic bacterium having a plant growth promoting effect, wherein the tea tree endophytic bacterium is Erwinia rhapontici (Erwinia rhapontici) V4 with a preservation number of CCTCC NO: m2021027.

Preferably, the tea plant endophytic bacterium has the 16S rDNA sequence of SEQ ID NO: 1, or a nucleotide sequence represented by the formula (I).

In a second aspect, the present invention provides a microbial preparation comprising the tea plant endophytic bacterium according to the first aspect of the present invention.

Preferably, the tea tree endophytic bacteria contained in the microbial inoculum are live bacteria, and the microbial inoculum is a liquid microbial inoculum.

In a third aspect of the invention, there is provided the use of the endophytic bacterium of the first aspect of the invention or the use of the microbial inoculum of the second aspect of the invention for promoting plant growth.

Preferably, the plant comprises tea or rice.

In a fourth aspect the invention provides a method of promoting the growth of tea plant comprising: contacting the tea plant endophyte according to the first aspect of the invention and/or the inoculant according to the second aspect of the invention with a tea plant seedling or plant.

Preferably, the method according to the fourth aspect of the present invention comprises the following specific steps:

(1) inoculating the tea tree endophytic bacteria to an LB liquid culture medium, carrying out shake culture to a logarithmic growth phase, centrifugally collecting the bacteria, cleaning with sterile water, and carrying out heavy suspension to obtain a microbial inoculum;

(2) and planting the tea tree seedlings or the tea tree plants in a sterilized nutrient medium, and inoculating the microbial inoculum for cultivation.

Preferably, in step (1), the shake culture conditions are 28 ℃ and 180 r/min.

Preferably, in step (1), the centrifugation time is 1min and the centrifugation speed is 10000 rpm.

Preferably, in step (2), the nutrient medium comprises: nutrient soil (Pindstrup substrate) with the mass portion ratio of 1: 1, vermiculite and perlite.

Preferably, in step (2), the microbial inoculum has a cell density of 1.0 at OD600 and an inoculum size of 50mL per strain.

In a fifth aspect, the present invention provides a method for promoting the growth of rice, comprising: contacting the tea plant endophyte according to the first aspect of the invention and/or the inoculant according to the second aspect of the invention with rice seeds.

Preferably, the method according to the fifth aspect of the present invention comprises the following specific steps:

(1) inoculating the tea tree endophytic bacteria to an LB liquid culture medium, carrying out shake culture to a logarithmic growth phase, centrifugally collecting the bacteria, washing with physiological saline, and carrying out heavy suspension to obtain a microbial inoculum;

(2) soaking the rice seeds in the day-soaking night dew until the rice seeds are exposed, and then sterilizing to obtain exposed white seeds with sterilized surfaces for later use;

(3) inoculating the leucorrhea seeds with the microbial inoculum, soaking, then cleaning the leucorrhea seeds with sterile water, and planting the leucorrhea seeds on a water culture medium for cultivation.

Preferably, in step (1), the shake culture conditions are 28 ℃ and 180 r/min.

Preferably, in step (1), the centrifugation time is 1min and the centrifugation speed is 10000 rpm.

Preferably, the step (A)2) The method specifically comprises the following steps: with 0.1% KMnO₄And (2) soaking and germinating the rice seeds for 24 hours in a day-night dew soaking mode, soaking and germinating the rice seeds for 24 hours in a day-night dew soaking mode by using sterile water until the rice seeds are exposed to the white, and then disinfecting the exposed white seeds for 3 minutes by using 75% alcohol and 5% sodium hypochlorite respectively to obtain the exposed white seeds with disinfected surfaces for later use.

Preferably, in the step (3), the bacterial concentration of the bacterial agent is 1.0 at OD 600; the common dew seeds are prepared according to the following steps of 1mL of microbial inoculum: the microbial inoculum is inoculated by 10 dewed white seeds.

Preferably, in step (3), the aqueous medium is 0.55% Agar (solid medium with only 0.55% Agar added to the water).

Different from the prior art, the technical scheme provides tea tree endophytic bacteria with a plant growth promoting effect, wherein the tea tree endophytic bacteria is Erwinia rhapontici (Erwinia rhapontici) V4, and the preservation number is CCTCC NO: m2021027, which has IAA producing ability, ACC deaminase activity, nitrogen fixing ability, phosphorus dissolving ability and siderophore producing ability, thereby improving soil nutrient components and absorption status, promoting plants to absorb more nutrients, and effectively promoting plant growth. Moreover, the tea tree endophytic bacteria are easy to culture, high in propagation speed, green, organic and pollution-free, and can be widely applied to preparation of plant growth promoting microbial agents or microbial fertilizers.

Drawings

FIG. 1 is a phylogenetic tree of Erwinia rhapontici V4 described in example 3;

FIG. 2 is a graph showing the results of measurements of tea tree growth indicators after inoculation of the experimental group (Erwinia rhapontici V4) and the control group (sterile water SW) described in example 4 (measurements after 13 weeks of inoculation);

FIG. 3 is a graph of the morphology of tea plants after inoculation of the experimental group (Erwinia rhapontici V4) and the control group (sterile water SW) as described in example 4 (photographs taken of the plants 13 weeks after inoculation);

FIG. 4 is a graph showing the results of measurement of rice growth indicators (measured after 14 days of culture) after inoculation of the experimental group (Erwinia rhapontici V4) and the control group (saline SNS) described in example 5, wherein a and b represent the level of significant difference; the same letter indicates that the difference between groups is not significant; the letters are different, and the difference between groups is significant;

FIG. 5 shows the morphology of rice plants inoculated in the experimental group (Erwinia rhapontici V4) and the control group (saline SNS) described in example 5 (photographs of the plants were taken after 14 days of culture).

Detailed Description

To explain technical contents, structural features, and objects and effects of the technical solutions in detail, the following detailed description is given with reference to the accompanying drawings in conjunction with the embodiments.

In the present invention, the formulation of the microbial inoculum (including Erwinia rhapontici V4 strain) is not particularly limited, and the microbial inoculum may be prepared into different formulations according to the intended use, and may be added with corresponding components such as excipients. For example, the microbial inoculum may be a liquid microbial inoculum (e.g., a bacterial solution) and/or a solid microbial inoculum (e.g., a lyophilized microbial inoculum), and is preferably a liquid microbial inoculum.

In the invention, the method of soaking the night dew in the day is to soak seeds in the day (10-12 h) and take out and spread the seeds in the night (10-12 h).

The experimental procedures in the following examples are conventional unless otherwise specified. The strains, plant crops, reagents, equipment and the like used in the following examples are commercially available unless otherwise specified. In the quantitative tests in the following examples, three replicates were set, and the data are the mean or the mean ± standard deviation of the three replicates.

First, the strains, culture media, kits and the like according to examples of the present invention will be described.

1. Description of the Strain preservation information:

the tea tree endophytic bacteria with the plant growth promoting effect provided by the invention is Erwinia rhapontici (Erwinia rhapontici) V4, and is preserved in China center for type culture Collection (CCTCC for short in preservation organization, the address is in China, Wuhan university) at 7 days 1 month 2021, the preservation number is CCTCC NO: m2021027.

2. Description of the medium composition:

(1) LB solid medium: 10g/L of tryptone, 10g/L of sodium chloride, 5g/L of yeast extract and 18g/L of agar;

(2) LB liquid medium: 10g/L of tryptone, 10g/L of sodium chloride and 5g/L of yeast extract;

(3) NA medium: 3g/L beef extract, 5.0g/L peptone, 2.5g/L glucose, 18g/L agar, pH 7.0(25 ℃);

(4) TSA tryptone soy agar medium purchased from Hangzhou microbial agents, Inc.

(5) DF solid culture Medium: KH (Perkin Elmer)₂PO₄4g/L，Na₂HPO₄6g/L，MgSO₄·7H₂O0.2 g/L, glucose 2g/L, gluconic acid 2g/L, citric acid 2g/L, (NH)₄)₂SO₄2g/L, agar 18g/L, pH 7.0(25 ℃);

(6) ADF solid medium: substitution of (NH) with ACC₄)₂SO₄As a unique nitrogen source, the final concentration is 3mmol/L, and other components are the same as DF solid medium;

(7) ashby solid medium: CaCO₃5g/L，CaSO₄.2H₂O0.1g/L，KH₂PO₄0.2g/L，MgSO₄·7H₂0.2g/L of O, 0.2g/L of NaCl, 10g/L of mannitol, 13g/L of agar, and 7.0 of pH (25 ℃);

(8) NBRIP solid Medium: glucose 10g/L, Ca₃(PO₄)25g/L，MgCl₂·7H₂O5g/L，(NH₄)₂SO₄0.1g/L，MgSO₄·7H₂O0.25g/L, KCl0.2g/L, agar 18g, pH 6.8-7.0(25 ℃);

(9) MKB solid medium: casein amino acid 5g/L, glycerin 15mL/L, KH₂PO₄2.5g/L，MgSO₄·7H₂o2.5g/L, agar 18g/L, pH 7.2(25 ℃);

(10) CAS solid medium: 60.5g of Chromoazuron S (CAS), 72.9mg of hexadecyltrimethylammonium bromide HDTMA, 1mmol/L, FeCl₃·6H₂O10mL, 50mL of 0.1mol/L phosphate buffer solution and 18g of agar, and metering to 1L; phosphorus (P)Acid salt buffer (g/L): NaH₂PO₄·2H₂O5.905g，Na₂HPO₄·12H₂O24.270g，NH₄Cl2.500g，KH₂PO₄0.750g, NaCl 1.250g, to volume of 1L, pH 6.8(25 ℃).

3. Description of the kit:

in the embodiment of the invention, the adopted bacterial genome extraction kit comprises: bacterial genome extraction kit from the name of the Yue organism Biomiga.

The present invention will be described in detail with reference to specific examples, but the present invention is not limited to the following embodiments.

Example 1 isolation and purification of tea Tree endophytic bacteria

Tea plant leaves are picked from the tea trees planted in Huangshan mountain of Anhui province, put into a clean sampling bag (bottle), marked and stored in a refrigerator at 4 ℃ for later use.

After the surfaces of the leaves of the Huangshan population tea trees are disinfected, cutting leaf samples into pieces, putting the pieces into a sterilized mortar, and adding a small amount of sterile water for grinding; then, the grinding fluid was transferred into a centrifuge tube by a tip, and sterile water equivalent to the mass of the blades was added to prepare a grinding fluid, which was then diluted into 3 concentration gradients 10^-1，10^-2，10^-3. Then, the three concentration-gradient slurries were applied to three solid media for specifically culturing bacteria, i.e., LB medium, NA medium, and TSA medium, respectively. After hardening, putting the coated plate into an incubator at 28 ℃ for 3 days, picking single colonies with different forms, repeatedly streaking for three times, purifying to obtain a pure culture, inoculating the pure culture to an LB solid culture medium test tube inclined plane, and storing at 4 ℃.

Example 2 screening of tea plant endophytic bacteria having plant growth promoting Effect

1. Culturing the strain and preparing a bacterial suspension:

the strain separated and purified in the embodiment 1 is inoculated into an LB liquid culture medium, shake cultivation is carried out for 8-12 hours under the conditions of 28 ℃ and 180r/min, the thalli are collected by centrifugation, and the thalli are washed by normal saline and are resuspended to prepare bacterial suspension.

2. Screening of strains

The separated strain is subjected to growth promotion effect identification through 5 plant growth promotion indexes of IAA production capacity, ACC deaminase activity, nitrogen fixation capacity, phosphorus dissolving capacity and siderophore production capacity, so that endophytic bacteria with plant growth promotion capacity are screened out.

(1) IAA Productivity test

Auxins (IAA) have a direct growth-stimulating effect on plants.

The testing steps are as follows: placing 1mL of the prepared bacterial suspension into a bacteria shaking tube containing 5mL of LB liquid culture medium (containing or not containing 0.5mg/mL of tryptophan) respectively, culturing for 2 days under the culture conditions of 28 ℃ and 180r/min, centrifuging the culture for 1min at the rotation speed of 10000rpm, taking 2mL of supernatant to react with 2mL of salkowski color developing solution, and storing for 30min in a dark place. In addition, 2mL of 60. mu.g/mL IAA sample, 2mL of LB liquid medium and 2mL of salkowski color developing solution were used as a control group. When the reaction solution became red, it was confirmed that the strain had IAA-producing ability.

(2) ACC deaminase activity assay

1-aminocyclopropane-1-carboxylic Acid (ACC) in plants is a precursor substance for ethylene synthesis, and an ACC deaminase-producing endophytic bacterium can degrade ACC into alpha-ketobutyrate and ammonia by using the ACC deaminase, so that the ethylene concentration under adverse conditions is reduced, the generation of harmful ethylene is reduced, and the rapid growth of plants is promoted.

The testing steps are as follows: 100 mu L of the prepared bacterial suspension is respectively coated on an ADF solid culture medium and a DF solid culture medium, and is cultured for 7 days in a constant temperature incubator at 28 ℃, and the growth condition on the DF solid culture medium is used as a control. The strain can still grow on an ADF solid medium after 5 generations of growth and subculture, and the strain has ACC deaminase synthesis capacity.

(3) Nitrogen fixation Capacity test

The testing steps are as follows: 100 mu L of the prepared bacterial suspension is respectively smeared on an Ashby solid culture medium, and a nitrogen-containing LB solid culture medium is used as a control. After the strain is cultured in an incubator at the constant temperature of 28 ℃ for 7 days, the strain can continue to grow on an Ashby solid medium, subculture is continued for 2 generations, and the strain is proved to be a positive strain and to have the nitrogen fixing capacity by the same characterization.

(4) Phosphorus dissolution test

The testing steps are as follows: the NBRIP solid medium was partitioned, 5. mu.L of the prepared bacterial suspension was spotted in each region to ensure colonies of the same size, and 3 media were spotted in each bacterial suspension (ensuring 3 replicates). After culturing for 7 days in an incubator at 28 ℃, a transparent halo is observed around the strain, indicating that the strain has phosphorus solubilizing ability.

(5) Test for siderophore Generation ability

The iron carrier can promote the absorption of plants to iron in soil and also can prevent and treat pathogenic bacteria in soil.

The testing steps are as follows: partitioning the MKB solid culture medium, planting 5 mu L of prepared bacterial suspension in each region, ensuring that the sizes of bacterial colonies are the same, and culturing in a constant-temperature incubator at 28 ℃. 3 medium were spotted per suspension (3 replicates were guaranteed). And after the strain grows up, cooling the heated CAS solid culture medium to 60 ℃, pouring 10mL of the heated CAS solid culture medium to uniformly cover the surface of the MKB solid culture medium containing the strain, and if an iron carrier halo is observed around the bacterial colony on the plate after 1 hour, indicating that the strain has the capability of generating the iron carrier.

Through the determination of the 5 plant growth promotion indexes, a tea tree endophytic bacterial strain with the best IAA production capacity, ACC deaminase activity, nitrogen fixation capacity, phosphorus dissolving capacity and siderophore production capacity is obtained through screening, and is named as V4.

Example 3 identification and preservation of endophytic bacterium of tea tree V4 having plant growth promoting Effect

1. Molecular identification of tea tree endophytic bacteria V4

The genomic DNA of the tea tree endophytic bacterium V4 described in example 2 was extracted using a bacterial genome extraction kit (zee bio Biomiga brand) and the 16S rDNA sequence of the strain was amplified using bacterial universal primers. Wherein, the base sequence of the bacterial universal primer is as follows:

F16S-27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (shown in SEQ ID NO: 2);

R16S-1492R: 5'-CGGTTACCTTGTTACGACTTC-3' (shown in SEQ ID NO: 3).

The amplified product was sequenced by the general biological systems (Anhui) Ltd, the length of the 16S rDNA sequence was 1443bp, and the sequence was as shown in SEQ ID NO: 1 is shown.

Homology analysis of the determined sequences with known nucleotide sequences using Blast software in NCBI database showed that the endophytic bacteria of tea tree strain V4 has the highest similarity (99.58%) with the 16S rDNA sequence of Erwinia rhapontici strain DQ-03(KF 500096.1).

Subsequently, the tea tree endophytic bacterium strain V4 and 5 strain sequences in Erwinia were subjected to cladode analysis, i.e., Mega5.0 software was used to perform multiple sequence matching arrangement, and a 16S rDNA phylogenetic tree was constructed by the Neighbor-Joining Method, and the analysis results are shown in fig. 1. As can be seen from FIG. 1, the endophytic bacterium strain V4 of Camellia sinensis is closest to Erwinia rhapontici, and can be identified as belonging to Erwinia. The endophytic bacterium strain V4 of the tea tree is identified as Erwinia rhapontici (Erwinia rhapontici V4) with the number of V4 by combining morphological characteristics and physiological and biochemical characteristics.

2. Preservation of endophytic bacteria of tea tree V4

The Erwinia rhapontici (Erwinia rhapontici V4) is preserved in China center for type culture Collection (CCTCC for short in the preservation unit, with the address of China, Wuhan university), and the preservation number is CCTCC NO: m2021027, preservation date 1/7/2021.

Example 4 application of endophytic bacterium V4 from tea tree to tea tree

Annual Longjing 43 cutting seedlings are used as experimental materials.

Washing the roots of the tea seedlings with clear water, removing original soil, scrubbing leaves with clear water, selecting 24 tea seedlings with the same size, pruning and keeping 20 cm lignified overground parts.

Tea seedlings are planted in sterilized nutrient media, the same amount of sterilized water is added into each pot of nutrient media, one tea seedling is planted in each pot, and the tea seedlings are cultured in a greenhouse (25 ℃, 75% humidity, 16 hours of light and 8 hours of dark) for 7 days for later use. Wherein the nutrient medium comprises nutrient soil, vermiculite and perlite with the mass portion ratio of 1: 1. Wherein the nutrient soil is peat (Pindstrup substrate) purchased from Topu group of Denmark.

Erwinia rhapontici V4 strain was inoculated into LB liquid medium, and shake-cultured at 28 ℃ and 180rpm to logarithmic phase. Subsequently, the cells were centrifuged at 10000rpm for 1min, washed with sterile water, and resuspended until the OD600nm became 1.0 (using an ultraviolet spectrophotometer) as an inoculum and the sterile water was used as a control (denoted as SW). 50mL of inoculation microbial inoculum is inoculated to each pot of tea seedlings in the experimental group, 50mL of sterilized water is inoculated to each pot of tea seedlings in the control group, and the inoculation is carried out once a week for 13 weeks.

In this example, the effective inoculation concentration of the microbial inoculum was 1.0 at OD600nm, and the inoculation amount was 50 mL. It can be understood by those skilled in the art that in other embodiments, the specific inoculation amount can be adjusted according to the change of the thallus concentration, and will not be described herein. After 13 weeks of inoculation, the growth index of the tea plant was measured: the young shoot germination rate, the young shoot length, the fresh weight and the dry weight of the young shoots, the number of leaves of the young shoots and the detection results are shown in table 1 and fig. 2. FIG. 3 is a graph showing the morphology of tea seedlings in the experimental group (Erwinia rhapontici V4) and the control group (sterile water SW) 13 weeks after inoculation.

TABLE 1 tea tree growth index test results

Note: a. b represents the level of significant difference; the same letter indicates that the difference between groups is not significant; letters different, indicating significant differences between groups.

As can be seen from table 1, fig. 2 and fig. 3, when the Erwinia rhapontici V4 strain was inoculated, the young shoot germination rate, the young shoot length, the young shoot fresh weight, the young shoot dry weight and the number of young shoot leaves of tea plant were significantly increased compared to the control group inoculated with sterilized water. The results show that Erwinia rhapontici V4 strain has obvious and effective growth promoting effect on tea tree.

Example 5 application of tea tree endophytic bacterium V4 to Rice

The Shuangyou 899 rice seeds were used as experimental material.

With 0.1% KMnO₄Soaking the rice seeds for 24 hours in a day-night dew soaking mode, and then soaking the rice seeds in the same day-night dew soaking mode by using sterilized water until the rice seeds are whitened. Then, carrying out the following steps; sterilizing the lubai seeds with 75% alcohol and 5% sodium hypochlorite for 3 minutes respectively to obtain surface-sterilized lubai seeds for later use.

Erwinia rhapontici V4 strain was inoculated into LB liquid medium, and shake-cultured at 28 ℃ and 180rpm to logarithmic phase. Subsequently, the cells were centrifuged at 10000rpm for 1min, collected, and resuspended in 0.9% physiological saline to an OD600nm of 1.0 (using an ultraviolet spectrophotometer) as an inoculum, and 0.9% physiological saline as a control (SNS). Then, 30 surface-sterilized white-exposed seeds were soaked in 3mL of an inoculating agent, inoculated at room temperature for 24 hours, washed with sterile water, planted on a 0.55% aqueous medium, and cultured in a greenhouse (25 ℃, 75% humidity, 16 hours light, 8 hours dark) for 14 days. Of these, 0.55% aqueous medium was 0.55% Agar (solid medium with only 0.55% Agar added to water).

In this example, the effective inoculation concentration of the microbial inoculum was 1.0 at OD600nm, and the inoculation ratios were: 1mL of a microbial inoculum: 10 dewed white seeds. It can be understood by those skilled in the art that in other embodiments, the specific inoculation ratio can be adjusted according to the change of the thallus concentration, and the details are not described herein.

After 14 days of culture, detecting the growth indexes of the rice seedlings: the seedling length, the number of roots, fresh weight, dry weight, and the results are shown in Table 2 and FIG. 4. FIG. 5 is a graph showing the morphology of rice seedlings in the experimental group (Erwinia rhapontici V4) and the control group (saline SNS) after 14 days of culture.

TABLE 2 Rice seedling growth index test results

Note: a. b represents the level of significant difference; the same letter indicates that the difference between groups is not significant; letters different, indicating significant differences between groups.

As can be seen from table 2, fig. 4 and fig. 5, the seedling length, the number of roots, the fresh weight and the dry weight of rice were significantly increased after inoculation of the Erwinia rhapontici V4 strain compared to the control group inoculated with 0.9% physiological saline, indicating that the Erwinia rhapontici V4 strain had a significant and effective growth promoting effect on rice.

The tea tree endophytic bacteria and the microbial inoculum provided by the invention have IAA generation capacity, ACC deaminase activity, nitrogen fixation capacity, phosphorus dissolving capacity and siderophore generation capacity, can improve soil nutrient components and absorption conditions, and promote plants to absorb more nutrients, thereby effectively promoting the growth of the plants. Therefore, the tea tree endophytic bacteria and the microbial inoculum provided by the invention are not limited to be applied to promoting the growth of tea trees and rice, and can also be applied to promoting the growth of other plants.

It should be noted that, although the above embodiments have been described herein, the invention is not limited thereto. Therefore, based on the innovative concepts of the present invention, the technical solutions of the present invention can be directly or indirectly applied to other related technical fields by making changes and modifications to the embodiments described herein, or by using equivalent structures or equivalent processes performed in the content of the present specification and the attached drawings, which are included in the scope of the present invention.

Sequence listing

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gcgtagagat ctggaggaat accggtggcg aaggcggccc cctggacaaa gactgacgct 720

caggtgcgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca cgccgtaaac 780

gatgtcgact tggaggttgt gcccttgagg cgtggcttcc ggagctaacg cgttaagtcg 840

accgcctggg gagtacggcc gcaaggttaa aactcaaatg aattgacggg ggcccgcaca 900

agcggtggag catgtggttt aattcgatgc aacgcgaaga accttacctg gccttgacat 960

ccacggaatt tggcagagat gccttagtgc cttcgggaac cgtgagacag gtgctgcatg 1020

gctgtcgtca gctcgtgttg tgaaatgttg ggttaagtcc cgcaacgagc gcaaccctta 1080

tcctttgttg ccagcacgta atggtgggaa ctcaaaggag actgccggtg ataaaccgga 1140

ggaaggtggg gatgacgtca agtcatcatg gcccttacgg ccagggctac acacgtgcta 1200

caatggcgca tacaaagaga agcgacctcg cgagagcaag cggacctcat aaagtgcgtc 1260

gtagtccgga tcggagtctg caactcgact ccgtgaagtc ggaatcgcta gtaatcgtag 1320

atcagaatgc tacggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccatgg 1380

gagtgggttg caaaagaagt aggtagctta accttcggga gggcgctacc acttgatcgt 1440

gcg 1443

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence (Artificial)

<400> 2

agagtttgat cctggctcag 20

<210> 3

<211> 21

<212> DNA

<213> Artificial sequence (Artificial)

<400> 3

cggttacctt gttacgactt c 21

Claims (10)

1. The tea tree endophytic bacterium with the plant growth promoting effect is characterized in that the tea tree endophytic bacterium is Erwinia rhapontici (Erwinia rhapontici) V4, and the preservation number is CCTCC NO: m2021027.

2. The endophytic bacterium of tea tree according to claim 1, wherein the 16S rDNA sequence of the endophytic bacterium of tea tree is SEQ ID NO: 1, or a nucleotide sequence represented by the formula (I).

3. An agent comprising the endophytic bacterium of Camellia sinensis as claimed in claim 1 or 2.

4. The microbial inoculum according to claim 3, wherein the tea plant endophyte contained in the microbial inoculum is a live bacterium, and the microbial inoculum is a liquid microbial inoculum.

5. Use of the endophytic bacterium of Camellia sinensis as claimed in claim 1 or 2, or the microbial agent as claimed in claim 3 or 4 for promoting plant growth.

6. Use according to claim 5, wherein the plant comprises tea or rice.

7. A method of promoting the growth of tea plants, comprising: contacting the endophytic bacterium of Camellia sinensis as claimed in claim 1 or 2 and/or the microbial inoculum of claim 3 or 4 with a young plant of Camellia sinensis or a plant of Camellia sinensis.

8. The method according to claim 7, characterized by comprising the following specific steps:

(1) inoculating the tea tree endophytic bacteria to an LB liquid culture medium, carrying out shake culture to a logarithmic growth phase, centrifugally collecting the bacteria, cleaning with sterile water, and carrying out heavy suspension to obtain a microbial inoculum;

(2) and planting the tea tree seedlings or the tea tree plants in a sterilized nutrient medium, and inoculating the microbial inoculum for cultivation.

9. A method of promoting the growth of rice, comprising: contacting the endophytic bacterium of Camellia sinensis as claimed in claim 1 or 2 and/or the microbial agent of claim 3 or 4 with rice seeds.

10. The method according to claim 9, characterized by comprising the following specific steps:

(1) inoculating the tea tree endophytic bacteria to an LB liquid culture medium, carrying out shake culture to a logarithmic growth phase, centrifugally collecting the bacteria, washing with physiological saline, and carrying out heavy suspension to obtain a microbial inoculum;

(2) soaking the rice seeds in the day-soaking night dew until the rice seeds are exposed, and then sterilizing to obtain exposed white seeds with sterilized surfaces for later use;

(3) inoculating the leucorrhea seeds with the microbial inoculum, soaking, then cleaning the leucorrhea seeds with sterile water, and planting the leucorrhea seeds on a water culture medium for cultivation.

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